Hydroperoxide diagnostic agents containing a chromogen indicator

ABSTRACT

D R A W I N G

United States Patent [72] Inventors Hans-Georg Rey;

Hans Wiellnger; Peter Rieckmann, all of Mannheim-Waldhof, Germany [2 l] App]. No. 21,494

[22] Filed Mar. 20, 1970 [45] Patented Dec. 14, 1971 [73] Assignee Boehringer Mannheim Gmbll Mannheim, Germany [32] Priorities Oct. 14, 1967 [3 3 Germany Apr. 9, 1969, Germany, No. P l9 17 996.5

Continuation-impart of application Ser. No.

762,965, Sept. 26, 1968. This application Mar. 20, 1970, Ser. No. 21,494

[54] IIYDROPEROXIDE DIAGNOSTIC AGENTS CONTAINING A CHROMOGEN INDICATOR 18 Claims, No Drawings [52] US. Cl 252/408, 23/230 B, 23/253 TP. 195/1035, 260/24! [51] Int. Cl G0ln 3l/l4. G0ln 31/22 [50] Field 0! Search 252/408; 23/230 B, 230 R, 253 TP; l95/l03.5; 424/7; 260/241 [56] References Cited UNITED STATES PATENTS 3,l83,l73 5/1965 Oakes 252/408 x Primary Examiner]0hn T. Goolkasian Assistant Examiner-M. E. McCamish Attorney-Burgess. Dinklage & Sprung ABSTRACT: 11, X

ROPERROXIDE DIAGNOSTIC AGENTS CONTG A CHROMOGEN INDICATOR This application is a continuation-in-part of Ser. No. 762,965 filed by Rey et al. on Sept. .26, 1968.

This invention relates to diagnostic agents for use in carrying out rapid analytical determinations and to methods for manufacturing and using such agents.

More particularly this invention relates to diagnostic agents for use in the analytical determination of hydroperoxides and of substances from which hydrogen peroxide and other hydroperoxides can be liberated by a previous reaction, as well as for the determination of peroxidase and other peroxidate-active substances.

The detection of glucose in urine, blood and serum has, in the case of diabetes, acquired considerable clinical importance as has the detection of peroxidase-active substances, such as hemoglobin in urine and blood, and the detection of hydroperoxides in, for example, the milk industry, the cosmetics industry and in polymer chemistry.

A series of compounds are known which are oxidized to dyestuffs by means of hydrogen peroxide and peroxidase as catalyst. Compounds of this type include, for example, the Nadi reagents, p-phenylene-diamine derivatives, carboazines, quaiac resin, aldazine and the like. Other compounds previously preferred for this purpose include for example benzidine, o-dianisidine, o-tolidine and guaiacol. However, it has been established that in practice certain of these compounds are not very stable and further according to recent findings, that they can also be dangerous to the health of the personnel working with them so that their use does not appear to be indicated in view of danger to personnel or reliability of results.

in accordance with the invention it has now surprisingly been found that compounds having the following formula:

wherein R, is lower alkyl; R, is hydrogen, a sulfonic acid group or an alkali metal sulfonate group; R is hydrogen, halogen, e.g. chlorine, or lower alkyl; X is sulfur, oxygen, a carbon atom substituted with two lower alkyl groups, substituted or unsubstituted vinylene or imino, wherein said substituent is lower alkyl or aryl; and Y is nitrogen or methine which, when taken together with R,, can form a benzene ring substituted by R,; are absolutely stable, physiologically safe and are particularly well suited for use in the determination of hydrogen peroxide, hydroperoxides, peroxidase and peroxidatively active substances carried out by means of colorimetry, optical tests and with test papers and test film.

It is known that compounds having the formula I as set out above can be oxidized by means of comparatively strong oxidation agents, such as lead tetraacetate, ceric sulfate and potassium persulfate, to provide colored oxidation products (cf. S. Hunig et al. Liebigs Ann. Chem, 676, 32-65/ 1964; H. Lang et al., Z. Analyt. Chem., 201, 321/1964). However, no reaction takes place in the presence of hydrogen peroxide alone. It was therefore not to have been expected that reproducible and clear colorations could also be obtained for hydrogen peroxide in the presence of peroxidase or of peroxidatively active substances.

According to a further feature of the present invention, it has now been found, that especially advantageous and clearly graduated color changes are realized when the compounds of formula I are used in admixture with reduction agents having the formula:

wherein Ar is benzene or naphthalene, V is hydroxyl or amino, V is hydrogen, hydroxyl or amino, which may be alkylated or arylated, W is amino, carboxy, a sulfonic acid group, alkyl, alkoxy or aryl and W is hydrogen, amino, carboxy or a sulfonic acid group, an alkali metal carboxylate group or sulfonate group, alkyl, alkoxy or aryl.

These compounds of formula I] do not themselves give any coloration or, at most, only give rise to very nonspecific colorations and are, therefore, generally not suitable per se for use as indicators. However, as already mentioned above, when these compounds of formula ii are used together with the compounds of formula I, there are realized particularly clear and well-graduated color changes.

Thus, according to the present invention, there is provided a process for the detemrination of hydroperoxides and of substances which react with the liberation of hydrogen peroxide, as well as of peroxidase and of peroxidatively active substances, comprising a chromogen which is oxidized by hydrogen peroxide or hydroperoxide in the presence of peroxidase or peroxidatively active substance to form a colored dyestufl', the color intensity of which can be evaluated in the conventional manner to indicate the quantity of hydrogen peroxide, hydroperoxide, peroxidase or peroxidatively active substance present in the specimen being analyzed wherein a compound of formula I is used as chromogen, preferably in admixture with a compound of formula ii.

The evaluation of the coloration can be carried out, for example, by optical measurement using therefor a spectrophotometer or, when test paper strips and test films are involved, by comparison of the color intensity with standard color scales or standard comparison solutions or with color charts.

The determination of the presence of hydroperoxides by the process according to the present invention is particularly useful for coupled and uncoupled enzyme reactions, as for example, for the determination of glucose, galactose, amino acids, uric acid, peroxides, hemoglobin, peroxidase or other peroxidatively active substances, as well as of enzyme activities. Because of the increasing importance of such determinations, the routine determination of substances of this type is now an essential feature of clinical chemistry and of foodstuff chemistry.

In the case of the determination of glucose, the latter is, for example, oxidized by glucose-oxidase to gluconic acid, atmospheric oxygen thereby being reduced to hydrogen peroxide. in the presence of peroxidase or of a peroxidatively active substance, the hydrogen peroxide then oxidizes the indicator or chromogen (formula I) used according to the present inventionto provide the corresponding color dyestuff.

yl-lactic acid.

According to a further feature of the present invention, there is provided a diagnostic agent for the determination of hydroperoxides and of substances which react with the liberation of hydrogen peroxide, which comprises peroxidase or a peroxidatively active substance and a chromogen having the formula I alone or in admixture with a compound of formula According to yet another feature of the present invention, there is provided a diagnostic agent for the determination of hemoglobin and of other peroxidatively active substances, which comprises hydrogen peroxide or a substance forming hydrogen peroxide and a chromogen of formula I alone or in admixture with a compound corresponding to formula II.

it is to be understood that, the context of the present invention, the expression a substance forming hydrogen peroxide" is intended to mean not only a single compound, such as an organic peroxide, but also a mixture, such as glucose and glucose-oxidase.

It is further to be understood that the new diagnostic agents according to the present invention can be prepared in the form of solutions in appropriate solvents, if necessary with the addition of conventional adjuvants, such as bufl'ers. Alternatively, the new diagnostic agents can be prepared in the form of test papers by the impregnation of suitable adsorbent materials, such as filter papers, with solutions of the components of the diagnostic reagents or such solutions can be used for making reagent films. In this connection, as in the case of the diagnostic solutions, it is frequently advantageous for the test papers and films to be prepared from solutions which contain conventional adjuvants, such as bufi'ers.

Finally, mention should be made of the fact that the process according to the present invention can, in addition, be used for the determination of the chromogens of formula I as well as of the compounds of formula ll, utilizing therefor a hydroperoxide and a peroxidate-active substance. This has proved to be very useful for control purposes in the preparation of these diagnostic materials.

The preparation of the compounds I can be carried out in known manner by the reaction of 2-haloor Z-rnercaptoquaternary salts with an appropriate N-alkylated hydrazone salt, with the addition of an amine, preferably of triethylamine, in a polar solvent, such as methanol, at a temperature of l5-l00 C. Depending upon the reactivity of the individual reaction components, the reaction is finished with a period of 10 minutes to 2 hours (S. Hiinig et al., Liebig's Ann. Chem., 676, 32-65/1964). The preparation of the hydrazones used as starting materials, as well as of the N-quatemizcd salts, takes place in the manner described by S. Hiinig Ann. Chem., 609, 160-180/1957) and by H. Balli (Liebigs Ann. Chem., 647, 1-8/1961).

A more comprehensive understanding of the invention may be obtained by referring to the following illustrative examples which are not intended, however, to be unduly limitative of the invention.

Example 1 Reagent Film for the Detection of Glucose 45 g. Propiofan (BASF), 35 g. of a solution of 37 g. Algipon in 2 liters of a 0.5M phosphate or citrate buffer having a pH of 5.7, l g. Texapon P (Henkel, Dusseldorf), 10 ml. water, 0.075 g. peroxidase and 0.15 g. glucose oxidase were stirred together to give a homogeneous slurry. To this mixture, there were added 6 ml. of a 5 percent aqueous solution of 2,2-azino-di-( 3 -ethylbenzthiazolone-6-sulfonic acid). Mixtures prepared in this manner, contain 0.2-0.3 percent of indicator. Foils were then coated with the mixture with a coating thickness of 300pand thereafter dried.

When glucose solutions of varying concentrations were evaluated with the above-described foils poorly differentiated green colorations were observed.

n the other hand, if the above-described mixture contained, in place of the aqueous solution of 2,2'- a zi no d i- (3- 5 in coloration.

However, if there was prepared a mixture analogous to the one described above but so it contained 6 ml. of an aqueous methanolic solution of a mixture of 5 percent 2,2 -azinodi- (3-ethyl-benzthiazolone-lS-sulfonic acid) and 5 percent p- 10 amino-salicylic acid, then using this mixture there could be prepared test strips which, depending upon the glucose concentration in the solutions tested produced colors in graduations from green to violet.

Example 2 Detection of Glucose in Blood, Urine or Serum Using a procedure analogous to that described in example I, reagent films were produced with the following compounds (I):

A. 2,2'-azino-di-( 3-ethyl-benzthiazolone-6-sulfonic acid) B. 2,2'-a.zinodi-( l-ethyl-quinoline-6-sulfonic acid) C. 2,2'-azino-di-( l,3-diethyl-benzimidazolone--sulfonic acid) D. l-[3-methyl-benzthiazolone-(2)]-2-[ l-ethyl-pyridone-(Z )]-azine E. l-[3-ethyl-benzthiazolone-(2)]-2-[ l-phenyl-3-methyl- 4- ethyll ,2,4-triazolone-( 5 ]-azine F. l-[3-ethyl-benzthiazolone-(2)]-2-[l-ethyl-quinolinone- (2)1-azine G. l-[3-methyl-benzthiazolone-(2)]-2-[ l-phenyl-S-methyl- 4-ethyl- 1 ,2,4-triazolone-( 5 l-azine H. l-[ l-ethyl-quinolinone-(2)]-2-[ lethyl-pyridone-(2)]- azine l. l-[3-ethyl-benztl'iiazolone-(2)]-2-[ l-ethyl-pyridone-(2)]- azzne J. l-[3-methyl-benzthiazolone-(2)]-2-[ l-ethyl-quinolinone- 2)]-azine K. l-[3-methyl-benzthiazolone-(2)] 2-[ l-ethyl-6- chloropyridone-(2)]-azine l... l-[ l-ethyl-quinolinone-( 2 ]-2-[ l-ethyl-6- chloropyridone-2)]-azine M. 2,2'-azino-di-( 3-ethyl-benzthiazolone) N. 2,2'-azino-di-( 3ethyl-benzoxazol-2-one) O. l-( 3-ethyl-benzoxazol-2-one )-2-( l-ethyl-pyrid-Z-one P. 1-( 3-ethyl-benzoxazol-2-one )-2-( 3-ethyl-benzthiazol-2- one)-azine Q. l-( 3-ethyl-benzoxazol-2-one )-2-( l-ethyl-quinoline-2- one)-azine R. l-( l-ethyl-3,3-dimethyl-indolinc-2-one)-2-( 3-ethylbenzthiazol-2one )-azine The above-mentioned compounds coming within the scope of formula I were used in combination with a number of different compounds within the scope of formula ll.

The resulting color changes produced when the corresponding reagent films were dipped into or moistened with various glucose-containing solutions, such as blood, urine, or serum, were evaluated after a short period of time. The specific details of the color reagents used and the color changes obtained therewith are summarized in the following table.

TABLE I Modifier (II) o-Arninophenol-p-sulfonic acid (l-amino naphthol-8)'2,4-disulfon8c acid. (7-amlnonaphthol-1) -3,6 llsodium sulionat Naphthyl-l-amine-B-sulfonlc acid Naphthyl-1-am1ne-8-sulfonlc acid m-Amino-phenol.

p-P henetidine Z-hydroxy--aminoW-methoxydlphenyl-arnino a-N aphtho1-2-sulfonlc acid p-Amlnosalicylic acid o-Amino-phenol Modifier cone. percent Dissolved in- Color reaction depending on glucose cone.

Green (graduated).

p-Amino-salicylic acid TABLE I Indicator Modifier cone. cone. Color reactlonde dl Indicator (I) percent Modifier (II) percent Dissolved inglucose conc. mu m on 01 o-Amino-phenol-p-sulfonic acid... 0.1 Water Yellow brown to violet.

01 (l-amino-naphthol-S)-2,44iisulfomc acid 0.1 do Grey-violet to blue. 01 Naphthyl-l-amine-o-sulfonic acid. 0.1 N/SO NaOH soln... Grey-violet. .1 0.1 Methanol Blue-red. .8 .3

p-Amino-salicylic acidp-Aminosallcyllc acid.

pAminosallcylic acid m-Aminophcnol O-Aminophenol-p-sulphonic acid (l-aminonaphthol-S)-2,4-disulphonic acid. p-Aminosalicylic acid m-Aminophenol p-Phenetidine.-. Naphthyl-l-smin ph Naphthyl-l-amine-8-su1phonic acid p-Aminosalicylio acid o-Arninophenol-p-su1ph0nic acid. (l-aminonaphthol-S)-2,4-disulphonic acid. p-Aminosalicylic acid p-Arninosalicylic acid m-Arninophcnol The percentages given in the above table refer to the whole mixture; the compounds (I) A-C were dissolved in water and D41 were dissolved in methanol.

Example 3 Detection of Blood in Urine Filter paper (Schleicher & Sch'ull No. 2316) was impregnated with a 0.1M phosphate buffer solution having a pH (l-amino-naphthol-S)-2,4-disulphonic acid. Naphthyl-l-amine-G-sulphonic acid (1-aminonaphtho1-8) -2,4-disulphonic acid of 7.0 in which 1 percent Texapon P (l-lenkel, Diisseldorf) had been dissolved and then the impregnated paper was dried. The dried paper was thereafier impregnated a second time with an aqueous solution of an indicator mixture (0.03 percent, 2,2- azinodi-[ l-ethylquinoline-6-sulfonic acid] and 0.1 percent o-aminophenolp-sulfonic acid) and dried.

When, to a paper than prepared, there was applied a drop of a blood-containing urine and a drop of 3 percent hydrogen peroxide solution then, even at a dilution of l:50,000, the paper became red-violet in color.

If, instead of o-amino-phenol-p-sulfonic acid there was used l-amino-8-naphthol -2,4-disulfonic acid, then, at a dilution of l:50,000, the paper became blue-violet in color.

Example 4 Detection of Hydroperoxide in Liquids a. Filter paper (Schleicher & Schtiil No. 2316) was impregnated with a 0.1M phosphate buffer solution having a pH of 6.0 and in which there had been dissolved, per mi. of bufier solution, 25 mg. peroxidase and 0.25 ml. Texapon P (Henltel, D'usseldorf). The impregnated paper was then dried. Thereafter, the paper was impregnated a further time using a solution of 0.01 g. 2,2-azino-di-( l-ethyl-quinoline-6-sulfonic acid) and 0.1 g. o-amino-phenol-p-sulfonic acid in 100 ml. water and the paper dried.

When there was applied to a paper thus prepared a drop of an aqueous hydropenoxide solution, then, even at a concentration of 0.2 y/ml. the paper assumeda red-violet coloration.

Green to dark green. Grey-violet to violet. Pink.

Rose to red-violet.

Lila

c. 1Igtosc to red-violet.

Methanol Lilac.

Pale to dark violet. Pale pink.

Pink to dark pink.

Grey-green.

.. Red-brown.

Yellow-green to green.

Yellow-green. Brown.

Do. Green. Grey-green to violet. Lilac.

Green.

. D0. Blue. Green. Pink. Do.

Red-brown. 0. 05 N/50 NaOH Flesh colored.

0. 1 Dirnethyl iormamide Pink violet. 0. l Methanol Orange 0. 2 0. 2

b. A filter pregnated with a solution of 40 mg. peroxidase and 0.25 ml. Texapon P in ml. of a 0.1M phosphate bufi'er of pH 5.6 and dried. The paper prepared in this manner was sub sequently impregnated with a cold, saturated solution in methanol of l-( 3-ethyl-benzoxazol-2-one )-2-( 3-ethylbenzthiazol- 2-one)-azine and dried. when solutions of hydrogen peroxide of various concentrations were applied to a reagent paper prepared in this manner, then the paper became green-blue, the strength of the color depending upon the hydrogen peroxide concentration. The lower-most detection limit was 0.01 'yhydrogen peroxide/ml.

c. One ml. of a 0.2 percent methanolic solution of Hiethyl-3,3-dimethyl-indolin-2-one)2 (3-ethyl-benzthiazol-2- one)-azine, 1 ml. of a 0.1 percent methanolic solution of pamino-salicylic acid, 0.05 ml. of a 1 percent solution of peroxidase in a 0.1M phosphate buffer of pH 5.7 and 0.05 ml. of a solution of hydrogen peroxide were pipetted together, stirred up and measured at a wavelength of 530 mm. The hydrogen peroxide concentration was determined with the help of a previously prepared calibrated curve.

Example 5 Detection of Glucose in Liquids 0.01 ml. of glucose solutions having concentrations of 0-300 mg.-percent were each pipetted into 2 ml. of an aqueous-methanolic 0.00125M indicator solution containing cquimolar amounts of 2,2'-azino-di-(3-ethyl-benzthiazolonefi-sulfonic acid) and p-amino-salicylic acid. There were then added to the resulting mixture 2 ml. of a solution of IO mg. peroxidase and 20 mg. glucose oxidase in 100 ml. of a 0.5M phosphate buffer, the pH of which was 5.7. Measurements were then carried out at a wavelength of 490 nm. and the corresponding glucose values calculated using therefor a calibratron curve.

When, as the indicator mixture, there was used 2,2'-azinodi-( l-ethyl-quinoline-6-sulfonic acid) and m-amino-phenol, the absorption maximum was established at 510 nm.

Example 6 at 300 milligram-percent blue-violet).

' The Texapon P used in the above examples is a detergentactive product. In place of Texapon P, there can be used, for

A reagent film was prepared using the procedures described exam 16 Sodium km 1 If 16 1h an I in example i, using an indicator mixture which contained 0.02 p ry Su a w] eq y good tspercent 2,2'-azino-di-( l-ethyl-quinoline-o-sulfonic acid) and 0.3 percent 2,2-azino-di-(3ethyl-benzthiazolone-6-sulfonic Example 9 acid). When these strips were employed in tests, as the glucose concentration increased, the color changed from violet to green. This color change could be very readily observed 10 Detection Glucose in Urine because the human eye is extremely sensitive to changes in A filt paper 23 f Schleicher & s was this $010 8 pregnated with a solution of 75 mg. peroxidase, 143 mg. glucose oxidase and 0.25 ml. Texapon P (l-lenkel, Dusseldorf) in g 7 100 ml. of a 0.1M phosphate bufier of pH 5.6 and then dried.

The paper impregnated in this manner was dipped into a solution of 0.2 g. l-( 3-ethyl-benzoxazol-2-one)-2-( l-ethyl-quin' Detection Glucose in Blood olin- 2-one)-azine and 0.1 g. p-aminosalicylic acid 100 ml.

methanol and dried.

blflod depi'oteiniled with 1 03M P Upon moistening a strip of a reagent paper produced in this acidof the supernatant liquid P p 5 20 manner with urine which contains a pathological amount of Of a 501mb" having the following composition: 1M glucose, there was obtained a red-violet color, the strength of phosphate'bufifer having a pH of 7.0 which contained per ml. hi h depends upon the glucose concentration.

40yg. peroxidase, 25073. glucose oxidase and 500m. of the diarnmonium salt of 2,2'-azino-di-(3-ethyl-benzthiazolone-6- sulfonic acid). The extinction was measured at a wavelength of 420 nm. and the corresponding glucose value read ofi from Example 10 a calibration curve.

Detection of Blood in Body Fluids E l 8 A filter paper (23 S of Schleicher & Sch'iill) was imxamp e W pregnated with a solution of 0.25 ml. Texapon P and 100 mg. sodium alginate in 100 ml. of a 0.1M phosphate bufi'er of pH Blood Glucose Test Strips 5.6 and dried. The paper thus prepared was then impregnated with a solution of 0.2 g. l-(3-ethyl-benzoxazol-2-one)-2-(1- Forty-five g. of an aqueous dispersion of polyvinyl ethyl-quinolin-2-one)-azine and 0.1 g. p-aminosalicylic acid in propionate (Propiofan, BASF), 35 g. of a solution of 37 g. 100 ml. methanol and dried.

sodium alginate (Algipon) in 2 liters of a 0.5M phosphate or When a drop of a body fluid which contains blood was apcitrate bufi'er having a pH of 5.7, l g. Texapon P (Henkel, plied to a reagent paper prepared in thismanner and a drop of Dusseldorf), 5 ml. water, 0.07 g. peroxidase and 0.16 g. glu- 3 percent hydrogen peroxide solution subsequently applied,

c'ose oxidase were stirred together to form a homogeneous 40 then the paper became red-violet-even at a blood concentraslurry. There were then added to this slurry 0.3 g. of the arm tion of 150,000.

monium salt of 2,2'-azinodi-(B-ethyI-benzthiazolone--sulfonic acid) and 0.04 g. of the ammonium salt of o-amino- Example 11 phenoLp-sulfonic acid dissolved in 7 ml. water, as well as 0.3

g. N,N,N', N'-tetramethyl-4,4'di-amino-diphenyhmethane dissolved in 4 ml. acetone. The mixture prepared in this i ofclucose m soluuons manner was applied, at a coating thickness of 3007, to a foil One ml. of a methanolic solution which contained one of and thereafter dried. the indicators (i) set out in the following table II in an amount When blood samples of varying glucose concentrations of up to 1 percent, depending upon the solubility, was mixed were applied to the thus prepared reagent films and the blood with 1 ml. of a solution of 150 mg. glucose oxidase and mg. left thereon for about 1 minute and thereafter wiped ofl", then, peroxidase in ml. of a 0.1M phosphate bufferof pH 5.6

in the physiological range, there were observed color graduaand 1 ml. of a glucose solution. There were obtained the color tions from yellow to green to blue. (Graduations: at 60 millireactions indicated in table II. As modifier, there was, in each gram-percent yellow; at milligram-percent green; at 180 55 case, used 0.1 ml. of a 1 percent methanolic solution of pmilligram-percent blue-green; at 240 milligram-percent blue; aminosalicylic acid.

TABLE II Indicator Color reaction compound M.P., designation Indicator compound name 0. Without modifier With modifier 2,2'-azin0-di-(3-ethyl-benzoxazol-2'ons) 216-217 Blue Red-brown. 1-(S-ethyl-benzoxazol-Zone)-2-(1-ethy1-pyrid-2-one)-azine 145446 Violet Red 1-(B-ethyl-benzoxazol-Z-one)-2-(1-ethyl-quinolln-2-one)-az1ne 199 do Red.

1-(3-ethy1-benzoxazol-2one)-2-(3-ethyl-benzthlazol-2-one)-azine 193-194 1-(3-ethyl-benzoxazo1-2-one)-2-(1,3-dlethyl-banzirnldazol-Q-one) -azine 215-216 1-(3Fethyl-benzoxazol-2one) -2-(Lethyl-fiehloropyridQ-one) -azlne 1-(1-ethyl-3,3-dimethyl-lndo1in-2one)-2-(3-ethylbenzthiazo1-2'one)-azine 208-209 1-(1-ethyl-B,3-dlmethyl-indolln-2-one) -2-(3-methylbenzthiazol-2-one) -azi .d 1-(1-ethyl-3,3-dimethyl-indo1ln-2-ona)-2-(lethyl-quinolinQ-one)-azine.

. 2,2'-azino-di-(3 ethyl-benzoxazol-2-one-6sulphonic acid) Red.

Brown-orange.

Red. Red-violet. D0.

Brown'orange. Redl-garown.

2,2-az1nodi'(3-ethyl-benzoxazol-2-one-6-sulphonlc acid) ammonium salt 0 0 1-( -ethyl-3,3-dirnethyl-lndolin-2-one)-2-(1-ethy1pyrid-2-one)-azlne Pale brown- 1-(1-ethyl-3,3-dimethyl-indo1in-2-one)-2-(1-ethyl-6-chloro-pyrld-2-one)-azine 146 1-(1-sthyl-8,8-dirnethyl-indoiin-2-ona)-2-(1,3-dlethylbenzlmidazol-2-one)-azine 177 Brown-oran 1-(1,3,3-trimethylindolin-2one)-2-(l-phenyl-3-methy1-4-ethyl-1,2,4-triazol-fi-one)-azin 244 Brown CC. 1-(1,3,3-trlmethyllndolln-2-one)-2-(3-ethyl-benzoxazol-Z-one)-azine 225 -H Y DD 1-(ii-ethyl-benzoxazol-2-one)-2-(1-phenyl-3-methyl-4-ethyl-1,2,4-trlazol-5-one)-azine 227 lDecomposltion. h V W W.

What is claimed is:

11. Diagnostic agent for use in the analytical determination of hydroperoxide, substances which react with the liberation of hydroperoxide, peroxidase and peroxidatively active substances comprising a first member selected from the group consisting of hydroperoxide, substances which react with the liberation of hydrogen peroxide, peroxidase and peroxidateactive substances and a second member comprising a chromogen of the formula:

wherein R, is lower alkyl, R, is a member selected from the group consisting of hydrogen, alkali metal sulfonate groups and sulfonic acid groups, R-, is a member selected from the group consisting of hydrogen, halogen and lower alkyl, X is a member selected from the group consisting of sulfur, oxygen, a carbon atom substituted with two lower alkyl groups, substituted and unsubstituted vinylene and imino, wherein said substituents are a member selected from the group of lower alkyl and aryl and Y is a member selected from the group consisting of nitrogen and methine, wherein said methine group member can be joined together with R to form a benzene ring substituted by R 2. Diagnostic agent according to claim 1 for use in the determination of hydroperoxide or a substance which reacts 4. Diagnostic agent as claimed in claim 1 wherein one, and i only one, of the X groups in the formula is oxygen or a carbon atom substituted with two lower alkyl groups.

5. Diagnostic agent as claimed in claim 1 wherein neither of the X groups in the formula is oxygen or a carbon atom substituted with two lower alkyl groups.

6. Diagnostic agent according to claim 1 in the form of a test paper impregnated with said diagnostic agent.

7. Diagnostic agent according to claim 1 in the form of a test film impregnated with said diagnostic agent.

8. Diagnostic agent according to claim 1 in the form of a solution thereof.

9. Diagnostic agent according to claim 1 additionally containing a compound having the formula:

.Jiliti.

wherein Ar is a member selected from the group consisting of bennene and naphthalene nuclei, V is a member selected from the group consisting of hydroxyl and amino, v is a member selected from the group consisting of hydrogen, hydroxy, amino, alkylated amino and arylated amino, W is a member selected from the group consisting of amino, carboxy, alkyl, alkoxy, aryl and sulfonic acid groups and W is a member selected from the group consisting of hydrogen, amino, carboxy, alkyl, alkoxy, aryl, sulfonic acid groups and alkali metal carboxylate or sulfonate groups, as modifier.

10. Diagnostic agent according to claim 2 additionally containing a compound having the formula:

wherein Ar is a member selected from the group consisting of benzene and naphthalene nuclei, V is a member selected from the group consisting of hydroxyl and amino, V' is a member selected from the group consisting of hydrogen, hydroxy, amino, allcylated amino and arylated amino, W is a member selected from the group consisting of amino, carboxy, alkyl, aryl, alkoxy and sulfonic acid groups and W is a member selected from the group consisting of hydrogen, amino, carboxy, alkyl alkoxy, aryl, sulfonic acid groups and alkali metal carboxylate or sulfonate groups, as modifier.

11. Diagnostic agent according to claim 3 additionally containing a compound having the formula:

wherein Ar is a member selected from the group consisting of benzene and naphthalene nuclei, V is a member selected from the group consisting of hydroxyl and amino, V' is a member selected from the group consisting of hydrogen, hydroxy amino, alkylated amino and arylated amino, W is a member selected from the group consisting of amino, carboxy, alkyl, alkoxy, aryl and sulfonic acid groups and W is a member selected from the group consisting of hydrogen, amino, carboxy, alkyl, alkoxy, aryl, sulfonic acid groups and alkali metal carboxylate or sulfonate groups, as modifier.

12. Diagnostic agent according to claim 1 wherein said chromogen (l) is 2,2'-azino-di-(3-ethyl-benzthiazolove-6-sulfonic acid).

13. Diagnostic agent according to claim 1 wherein said chromogen (l) is 2,2-azino'di-[l-ethylquinoline-o-sulfonicacid].

l4l. Diagnostic agent according to claim 9 wherein said chromogen (l) is 2,2'-azino-di-[l-ethyl-quinoline-6-sulfonic acid] and said modifier is o-amino-phenol-p-sulfonic acid.

15. Diagnostic agent according to claim 9 wherein said chromogen (I) is 2,2'-azino-di-(3-ethyl-benzthiazolone-6-sulfonic acid) and said modifier is p-amino-salicylic acid.

16. Diagnostic agent according to claim 9 wherein said modifier is a member selected from the group consisting of 0' amino-phenol-p-sulfonic acid, (1-amino-naphthol-8)-2, 4- disulfonic acid, (7-amino-naphthol-l )-3,6-disodium sulfonate, naphthyll -amino-6-sulfonic acid, naphthyll -amino8-sulfonic acid, p-aminosalicylic acid, o-amino-phenol, m-aminophenol, p-phenetidene, 2-hydroxy-4-amino-4-methoxydiphenylamine, and a-naphthol-Z-sulfonic acid.

17. Process for the analytical determination of hydroperoxide, a substance which reacts with the liberation of hydroperoxide, peroxidase and a peroxidate-active substance which comprises contacting a liquid containing the substance of interest with a reagent comprising a diagnostic agent according to claim 1, producing a color with respect to said 1 liquid as visual evidence of the presence of said substance of interest in said liquid.

18. Process for the analytical determination of hydroperoxide, a substance which reacts with the liberation of hydroperoxide, peroxidase and a peroxidate-active substance which comprises contacting a liquid containing the substance of interest with a reagent comprising a diagnostic agent according to claim 9, producing a color with respect to said liquid as visual evidence of the presence of said substance of interest in said liquid.

2 UNITED STIAIHPZS lA'll'lN'l' OFFICE 5 C9 1 1 q a \I e enn'rmcm 1; 01' CORRLQHON Patent No. 3,627,698 L Dated December 14, 1971 Inventor) Hans-Georg Rey, Hans Wielinger, Peter Rieckmann is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

ABSTRACT OF THE DISCLOSURE Diagnostic agents suitable for use in carrying out rapid analytical determinations of the presence and/or concentration of hydroperoxides, substances which react with the liberation of hydrogen peroxide or hydroperoxide,

peroxidase or peroxidatively active substances, comprising an indicator, iie. chromogen, which is oxidized by hydrogen peroxide or hydroperoxide in the presence of peroxidase or percxidatc active substai'lce to form a dyestuff, the color r I intensity of which is dependent on the quantity of peroxide, I peroxidase or peroxidate active substance present in the test sample, wherein the chromogen is a compound having the formula:

wherein R is lower alkyl: R is hydrogen, a sulfonic acid group or an alkali metal sulfonate group; R is hydrogen.

halogen, e.g. chlorine, or lower alkyl; X is sulfur, oxygen,

a carbon atom substituted with two lower alkyl groups, sub- ,atituted or unsubstituted vinylene or imino, wherein said eubstituent is lower alkyl or aryl: and is nitrogen or g methine which, when taken together with R can form a benzene ring substituted by R a4 .1 H a w UNITED STA'I'ISS PM, 1'11 1 01' HCE 4 CERIIFLCAIE OF CORRECTION 3,627,698 Dated December 14, 1971 Patent No.

Hans-Georg Rey, Hans Wielinger, Peter Rieehmann Inventor(s) If is certified that error appears in the above-identified patent and thin: said Letters Patent are hereby corrected as shown below:

' PAGE 2 col. 6,. Table 1, last columi'l, 3rd itein fr'm bottofi:

"L he" .should be "Lil o Signed and eeeled this 11 th day of July 19 72.

( SEAL) Attest ROBERT 'GOTTSCHALK Commissionerf of Patents EDL'JARD I-LFLETCHIFR, J1? Attesting Officer 

2. Diagnostic agent according to claim 1 for use in the determination of hydroperoxide or a substance which reacts with the liberation of hydrogen peroxide comprising a member selected from the group consisting of peroxidase and a peroxidatively active substance and a chromogen as defined in claim
 1. 3. Diagnostic agent according to claim 1 for use in the determination of peroxidatively active substances comprising a member selected from the group consisting of hydrogen peroxide and substances forming hydrogen peroxide and a chromogen as defined in claim
 1. 4. Diagnostic agent as claimed in claim 1 wherein one, and only one, of the X groups in the formula is oxygen or a carbon atom substituted with two lower alkyl groups.
 5. Diagnostic agent as claimed in claim 1 wherein neither of the X groups in the formula is oxygen or a carbon atom substituted with two lower alkyl groups.
 6. Diagnostic agent according to claim 1 in the form of a test paper impregnated with said diagnostic agent.
 7. Diagnostic agent according to claim 1 in the form of a test film impregnated with said diagnostic agent.
 8. Diagnostic agent according to claim 1 in the form of a solution thereof.
 9. Diagnostic agent according to claim 1 additionally containing a compound having the formula: wherein Ar is a member selected from the group consisting of benzene and naphthalene nuclei, V is a member selected from the group consisting of hydroxyl and amino, v'' is a member selected from the group consisting of hydrogen, hydroxy, Amino, alkylated amino and arylated amino, W is a member selected from the group consisting of amino, carboxy, alkyl, alkoxy, aryl and sulfonic acid groups and W'' is a member selected from the group consisting of hydrogen, amino, carboxy, alkyl, alkoxy, aryl, sulfonic acid groups and alkali metal carboxylate or sulfonate groups, as modifier.
 10. Diagnostic agent according to claim 2 additionally containing a compound having the formula: wherein Ar is a member selected from the group consisting of benzene and naphthalene nuclei, V is a member selected from the group consisting of hydroxyl and amino, V'' is a member selected from the group consisting of hydrogen, hydroxy, amino, alkylated amino and arylated amino, W is a member selected from the group consisting of amino, carboxy, alkyl, aryl, alkoxy and sulfonic acid groups and W'' is a member selected from the group consisting of hydrogen, amino, carboxy, alkyl alkoxy, aryl, sulfonic acid groups and alkali metal carboxylate or sulfonate groups, as modifier.
 11. Diagnostic agent according to claim 3 additionally containing a compound having the formula: wherein Ar is a member selected from the group consisting of benzene and naphthalene nuclei, V is a member selected from the group consisting of hydroxyl and amino, V'' is a member selected from the group consisting of hydrogen, hydroxy, amino, alkylated amino and arylated amino, W is a member selected from the group consisting of amino, carboxy, alkyl, alkoxy, aryl and sulfonic acid groups and W'' is a member selected from the group consisting of hydrogen, amino, carboxy, alkyl, alkoxy, aryl, sulfonic acid groups and alkali metal carboxylate or sulfonate groups, as modifier.
 12. Diagnostic agent according to claim 1 wherein said chromogen (I) is 2,2''-azino-di-(3-ethyl-benzthiazolone-6-sulfonic acid).
 13. Diagnostic agent according to claim 1 wherein said chromogen (I) is 2,2''-azino-di-(1-ethyl-quinoline-6-sulfonic-acid).
 14. Diagnostic agent according to claim 9 wherein said chromogen (I) is 2,2''-azino-di-(1-ethyl-quinoline-6-sulfonic acid) and said modifier is o-amino-phenol-p-sulfonic acid.
 15. Diagnostic agent according to claim 9 wherein said chromogen (I) is 2,2''-azino-di-(3-ethyl-benzthiazolone-6-sulfonic acid) and said modifier is p-amino-salicylic acid.
 16. Diagnostic agent according to claim 9 wherein said modifier is a member selected from the group consisting of o-amino-phenol-p-sulfonic acid, (1-amino-naphthol-8)-2, 4-disulfonic acid, (7-amino-naphthol-1)-3,6-disodium sulfonate, naphthyl-1-amino-6-sulfonic acid, naphthyl-1-amino-8-sulfonic acid, p-aminosalicylic acid, o-amino-phenol, m-amino-phenol, p-phenetidene, 2-hydroxy-4-amino-4-methoxy-diphenylamine, and Alpha -naphthol-2-sulfonic acid.
 17. Process for the analytical determination of hydroperoxide, a substance which reacts with the liberation of hydroperoxide, peroxidase and a peroxidate-active substance which comprises contacting a liquid containing the substance of interest with a reagent comprising a diagnostic agent according to claim 1, producing a color with respect to said liquid as visual evidence of the presence of said substance of interest in said liquid.
 18. Process for the analytical determination of hydroperoxide, a substance which reacts with the liberation of hydroperoxide, peroxidase and a peroxidate-active substance which comprises contacting a liquid containing the substance of interest with a reagent comprising a diagnostic agent according to claim 9, producing a color with respect to said liquid as visual evidence of the presence of said substance of interest in said liquid. 